TNF-α production inhibitor comprising kavalactone as an active ingredient

ABSTRACT

The present invention provides a TNF-α production inhibitor containing a kavalactone as an active ingredient, which inhibitor has high safety, exerts an excellent effect of inhibiting TNF-α production, and is useful as a drug or an animal drug for preventing, ameliorating, or treating diseases such as cachexia attributed to cancer or infectious diseases, chronic rheumatoid arthritis, inflammatory diseases, osteoarthritis, systemic lupus erythematosus (SLE), rejection during bone marrow transplantation, multiple organ failure, AIDS, meningitis, hepatitis, and type-II diabetes. The present invention also provides a preventive, ameliorating, or therapeutic agent for diseases caused by abnormal production of TNF-α, the agent containing a kavalactone as an active ingredient.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a TNF-α production inhibitor containinga kavalactone as an active ingredient, and to a preventive,ameliorating, or therapeutic agent for diseases caused by abnormalproduction of TNF-α.

2. Background Art

TNF (Tumor Necrosis Factor) was discovered as an antitumor cytokine, andhas been elucidated to have carcinostatic activity (i.e., the effect ofinhibiting cancer cell growth or necrotizing cancer cells), and toparticipate in a series of inflammatory responses or immunoreactions, aswell as in differentiation or maturation of cells.

Recent studies have shown that excessive production of TNF-α inducesonset of a variety of diseases, including cachexia attributed to canceror infectious diseases (Nature, 316: 552, 1985), septic shock (J.Immunol., 145: 4185, 1990; Science, 229: 869, 1985; Shock, 30: 1990),chronic rheumatoid arthritis (Ann. Rheum. Dis., 49: 665, 1990; Lancet,344: 1105, 1994; Lancet, 344: 1125, 1994; British J. Rheum., 34: 334,1995), inflammatory diseases such as ulcerative colitis and Crohndisease (Arch. Dis. Child, 66: 561, 1991; Gastroenterology),osteoarthritis (Arthritis Rheum., 36: 819, 1993), Kawasaki's disease(Clin. Immunol. Immunopathol., 56: 29, 1990), multiple sclerosis (N.Engl. J. Med. , 325(7): 467, 1991), Behchet's disease (J. Rheumatol. ,17: 1107, 1990), systemic lupus erythematosus (STE) (Arthritis Rheum.,32: 146, 1989), rejection during bone marrow transplantation (J. Exp.Med., 175: 405, 1992), multiple organ failure (Rinshoi, 17(20), 2006,1991), malaria (Science, 237: 1210, 1987), AIDS (J. Acquir. ImmuneDefic. Syndr., 5: 1099, 1992), meningitis (Lancet, 1: 355, 1987),hepatitis (Kozo Kanno, Kanzo, 33: 213, 1992), and type-II diabetes(Science, 259: 87, 1993).

The aforementioned diseases caused by excessive production of TNF-α havehitherto been treated from a mere palliative approach by use of steroidagents, anti-inflammatory agents, antibiotics, etc., and drugs forfundamentally treating the diseases have not yet been developed.

Kava is a plant found in Fiji and belongs to Piperaceae, Piper L.(nomenclature: Piper Methysticum Forst., alias: Yangona). Sinceanesthetic beverages are obtained from the kava root, in Oceania, kavais widely cultivated by privileged people and is used in traditionalceremonies or events (Chem. Australia. Oct 377–378 (1987)).

It has been reported that an extract obtained from the dried kava rootthrough extraction with water contains a class of α-pyrone derivativescalled kavalactones which induce numbness of the lips or tongue or exertsedative effect, such as methysticin (Chem. Australia. Oct 377–378(1987), Planta Med. 64 504–506 (1998)).

Studies performed In the University of New South Wales have elucidatedthat kavalactones exert a sedative effect through a mechanism differentfrom those of other sedative drugs which exert sedative effects whenbeing bound to receptors present in the brain (Planta Med. 65 507–510(1998)). It has also been reported that kavalactones exert an analgesiceffect in a manner different from that of a formulated analgesic drugsuch as aspirin, and that, unlike morphine, kavalactones are not boundto receptors in the brain (e.g., European Patent Application Laid-OpenNos. 664131 and 523591, and Japanese Kohyo (PCT) Patent Publication No.5-502457).

It has also been reported that kava extract exerts an antibacterialeffect and is useful for treating Helicobacter pylori infection (GermanPatent Application Laid-Open No. 19716660), and that the kava extractexerts a neuroprotective effect and is useful for treating braindysfunction, Alzheimer's disease, brain injury, etc. (e.g., EuropeanPatent Application Laid-Open No. 523591, and Japanese Kohyo (PCT) PatentPublication No. 5-502457).

However, until the present invention was attained, kavalactones and kavaextract have not been known to exert the effect of inhibiting TNF-αproduction.

SUMMARY OF THE INVENTION

In view of the foregoing, the present inventors have performed studieson naturally occurring substances which inhibit production of TNF-α, andhave found that kavalactones contained in kava extract exert anexcellent effect of inhibiting TNF-α production, and that thekavalactones are useful as TNF-α production inhibitors and aspreventive, ameliorating, or therapeutic agents for a variety ofdiseases caused by abnormal production of TNF-α. The present inventionhas been accomplished on the basis of this finding.

Thus, an object of the present invention is to provide a drug which isendowed with high safety, inhibits TNF-α production, and is useful as apreventive or therapeutic agent for the aforementioned diseases.

Accordingly, the present invention provides a TNF-α production inhibitorcomprising a kavalactone as an active ingredient.

The present invention also provides a preventive, ameliorating, ortherapeutic agent comprising a kavalactone as an active ingredient fordiseases caused by abnormal production of TNF-α.

The present invention further provides a method for the treatment ofdiseases caused by abnormal production of TNF-α, which method comprisesadministering an effective amount of a kavalactone.

The present invention further provides use of a kavalactone for themanufacture of a TNF-α production inhibitor.

Still, the present invention provides use of a kavalactone for themanufacture of a medicament for preventing, ameliorating, or treatingdiseases caused by abnormal production of TNF-α.

Preferably, the kavalactone is one or more species selected from thegroup consisting of desmethoxyyangonin, dihydrokavain, kavain, yangonin,methysticin, dihydromethysticin, and 7,8-epoxyyangonin.

BRIEF DESCRIPTION OF THE DRAWINGS

Various other objects, features, and many of the attendant advantages ofthe present invention will be readily appreciated as the same becomesbetter understood with reference to the following detailed descriptionof the preferred embodiments when considered in connection withaccompanying drawings, in which:

FIG. 1 shows a graph indicating inhibitory effects of KAVA-1, KAVA-2,KAVA-3, KAVA-4, and KAVA-5 on TNF-α production in BALB/3T3 cellsstimulated by okadaic acid; and

FIG. 2 shows a graph showing investigation results of inhibitory effectsof KAVA-1, KAVA-2, KAVA-3, KAVA-4, and KAVA-5 on TNF-α production inserum samples, the TNF-α production having been induced byintraperitoneally administering KAVA-1, KAVA-2, KAVA-3, KAVA-4, andKAVA-5 to BALB/c mice, followed by administration of LPS immediatelythereafter.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Kavalactone contained, as an active ingredient, in the TNF-α productioninhibitor of the present invention refers to a class of α-pyronederivatives contained in the root of kava (Piper Methysticum G. Forst)which belongs to Piperaceae, Piper L. Specific examples of the α-pyronederivatives include desmethoxyyangonin, dihydrokavain, kavain, yangonin,methysticin, dihydromethysticin, and 7,8-epoxyyangonin, which arerepresented by the following formulas.

These α-pyrone derivatives have a variety of isomers, includinggeometrical isomers such as cis-isomers and trans-isomers, opticalisomers such as d-isomers and 1-isomers, and rotational isomers. In thepresent invention, any of such isomers can be used, so long as theisomer can exert the effect of inhibiting TNF-α production. In thepresent invention, these pyrone derivatives also include racemicmodifications and a mixture of diastereomers.

From the viewpoint of the effect of inhibiting TNF-α production,examples of particularly preferred kavalactones includedesmethoxyyangonin (KAVA-3: compound 1), (+)-dihydrokavain (KAVA-4:compound 2), (+)-kavain (KAVA-5: compound 3), yangonin (KAVA-2: compound4), (+)-methysticin (KAVA-1: compound 5), (+)-dihydromethysticin(KAVA-6: compound 6), and 7,8-epoxyyangonin (compound 7).

These α-pyrone derivatives may be used singly or in combination of twoor more species as kavalactone employed in the TNF-α productioninhibitor of the present invention.

Kavalactones used in the present invention may be obtained from the kavaroot by means of a known extraction method, or obtained throughsynthesis by means of a published method (Acta Chemica Scandinavica B30, 7: 613–678, 1976; Planta Med., 64: 504, 1998).

In the case in which kavalactones are obtained through extraction, theaforementioned kavalactones separated from kava extract and purified canbe used. However, an extraction fraction containing a plurality ofcompounds may also be used, so long as the fraction exhibits the effectof inhibiting TNF-α production.

Since the thus-obtained kavalactones exert an excellent effect ofinhibiting TNF-α production in vivo and in vitro as described below inExamples, the kavalactones can be used as a TNF-α production inhibitorfor mammals including humans, and as a preventive, ameliorating, ortherapeutic agent for a variety of diseases caused by abnormalproduction of TNF-α, including cachexia attributed to cancer orinfectious diseases, septic shock, chronic rheumatoid arthritis,inflammatory diseases such as ulcerative colitis and Crohn disease,osteoarthritis, Kawasaki's disease, multiple sclerosis, Behchet'sdisease, systemic lupus erythematosus (SLE), rejection during bonemarrow transplantation, multiple organ failure, malaria, AIDS,meningitis, hepatitis, and type-II diabetes.

When the kavalactone according to the present invention is administeredas a drug, the amount and frequency of administration vary withpathological conditions, age, weight, manner of administration, andother conditions. In the case of peroral administration, the daily doseis typically 0.1–1,000 mg for adults, but the daily dose varies withpathological conditions and other conditions (e.g., 10–500 mg or 30–300mg). Regarding the case in which pure kavalactone is employed, a dailydose of 200 mg has been reported (Kretschmer “Kavain alsPsychopharmkon,” NMW 4/1970, 154–158).

When the kavalactone according to the present invention is used as aTNF-α production inhibitor and as a preventive, ameliorating, ortherapeutic agent for diseases caused by abnormal production of TNF-α,the kavalactone is prepared in the form of a typical pharmaceuticalproduct. For example, the kavalactone is formulated in a form suitablefor oral administration or parenteral administration (e.g.,intraarticular administration or enteric administration), such as apharmaceutical composition obtained by mixing the drug of the presentinvention with a pharmaceutically acceptable carrier (e.g., anexcipient, a binder, a disintegrant, a sweetening agent, a flavoringagent, an emulsifying agent, a diluent, or a dissolution promoter) andprocessing to have a product form of, for example, tablet, pill, powder,granule, capsule, troche, syrup, solution, emulsion, suspension, orinjection.

Examples of excipients include lactose, cornstarch sucrose, glucose,sorbitol, and crystalline cellulose. Examples of binders includepolyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose,gum arabi, tragacanth, gelatin, shellac, hydroxypropyl cellulose,hydroxypropyl starch, and polyvinyl pyrrolidone.

Examples of disintegrants include starch, agar, gelatin powder,crystalline cellulose, calcium carbonate, sodium hydrogencarbonate,calcium citrate, dextran, and pectin. Examples of lubricants includemagnesium stearate, talc, polyethylene glycol, silica, and hydrogenatedvegetable oil. Any pharmaceutically acceptable coloring agent may beused. Examples of sweetening and flavoring agents include cocoa powder,menthol, aromatic acids, peppermint oil, borneol, and cinnamon powder.If necessary, tablets or granules may optionally be subjected to sugarcoating, gelatin coating, or similar coating.

In the case of preparation of injections, if necessary, a pH-regulatingagent, a buffer, a stabilizer, or a preservative is added to theinjections, to thereby prepare agents for subcutaneous injection,intramuscular injection, or intravenous injection, by means of acustomary method. Injection preparations may be stored in a containerand freeze-dried, to thereby provide solid products, and the solidproducts may be prepared into injections upon use. A single dose of theinjection may be stored in a container, or a plurality of doses may bestored in a single container.

EXAMPLES

The present invention will next be described in more detail by way ofexamples, which should not be construed as limiting the inventionthereto.

Example 1 Isolation of Components Having a TNF-α Release InhibitoryEffect through Extraction

Dried kava root (1.0 kg) was subjected to extraction by use of methanolfor 16 days. The resultant mixture was subjected to filtration underreduced pressure, and the filtrate was concentrated under reducedpressure, to thereby yield a residue (152.0 g). The entirety of theresidue was partitioned by use of water and ethyl acetate (AcOEt), andthe resultant ethyl acetate phase was concentrated under reducedpressure, to thereby yield an ethyl acetate extract (76.17 g). Theentirety of the extract was added to a silica gel column (product ofMerck, 1 kg (70–230 mesh 500 g+230–400 mesh 500 g)) for chromatographyby use of n-hexane/ethyl acetate. In the course of chromatography, theethyl acetate concentration of the solvent was gradually elevated so asto effect elution/extraction in the following concentration profile:n-hexane (500 ml); 5% ethyl acetate/n-hexane (500 ml); 10% ethylacetate/n-hexane (500 ml); 15% ethyl acetate/n-hexane (500 ml); 20%ethyl acetate/n-hexane (500 ml); 25% ethyl acetate/n-hexane (500 ml);30% ethyl acetate/n-hexane (500 ml); 35% ethyl acetate/n-hexane (500ml); 40% ethyl acetate/n-hexane (500 ml); 45% ethyl acetate/n-hexane(500 ml); 50% ethyl acetate/n-hexane (500 ml); 60% ethylacetate/n-hexane (500 ml); 70% ethyl acetate/n-hexane (500 ml); 80%ethyl acetate/n-hexane (500 ml); 85% ethyl acetate/n-hexane (500 ml);90% ethyl acetate/n-hexane (500 ml); and ethyl acetate (500 ml).Collection of fractions was initiated from the eluate corresponding to20% ethyl acetate/n-hexane, with the volume of each fraction being 20ml. The eluate corresponding to fractions 65 to 74 was concentratedunder reduced pressure, to thereby yield 1.48 g of crude crystals. Thecrystals were recrystallized from ethyl acetate/n-hexane, to therebyyield 1.218 g of compound 1: 5,6-dehydrokavain, as pale yellow needles.In a similar manner, crude crystals (3.78 g) obtained from the eluatecorresponding to fractions 75 to 82 were recrystallized from ethylacetate/n-hexane, to thereby yield 3.513 g of compound 2: dihydrokavain,as colorless needles. Crude crystals (5.36 g) obtained from the eluatecorresponding to fractions 86 to 94 were recrystallized from ethylacetate/ether, to thereby yield 4.744 g of compound 3: kavain, ascolorless prisms. Crude crystals (1.27 g) obtained from the eluatecorresponding to fractions 97 to 102 were recrystallized from ethylacetate/ether, to thereby yield 0.847 g of compound 4: yangonin, as paleyellow prisms. The eluent corresponding to fractions 103 to 115 wasconcentrated under reduced pressure, to thereby yield a residue (8.21g). The residue was subjected to chromatography by use of a silica gelcolumn (product of Merck, 300 g (70–230 mesh 150 g+230–400 mesh 150 g))and chloroform/ether solvent. In the course of chromatography, the etherconcentration was gradually elevated. From the eluent corresponding to25% ether/chloroform, 1.322 g of compound 4: yangonin was obtained.Subsequently, crude crystals obtained from the eluent corresponding to30% ether/chloroform was recrystallized from ethyl acetate/ether, tothereby yield 5.661 g of compound 5: methysticin, as colorless needles.A residue (385 mg) obtained from the eluent corresponding to fractions138 to 142 was placed on a Sephadex LH-20 column (product of Pharmacia,30 g), and chromatography was performed by use of chloroform/methanol(1:1) solvent for development. Twenty mL of the eluent was collected perfraction (Fr.). From the eluent corresponding to fractions 5 to 7, 59 mgof pale yellow-white novel compound 7: 7,8-epoxyyangonin was obtained.

Compound 1 (KAVA-3) 5,6-Dehydrokavain (Desmethoxyyangonin);(4-methoxy-6-(2-phenylvinyl-2H-pyran-2-one):

m.p. 138–140° C., pale yellow needles EI-MS: m/z 228 (M⁺, 100%), 211(10%), 200 (36%), 185 (15%), 157 (27%) HR-MS: m/z 228.0763, C₁₄H₁₂O₃requires 228.0787, FT-IR (KBr) ν_(max) cm⁻¹: 3081, 1721 (C═O), 1644(C═C), 1611, 1557, 1256, 1154, UV (EtOH) λ_(max) nm (log ε): 344.5(4.32), 255 (4.05), 231.5 (4.15), 225 (4.14), 209 (4.28) 600 MHz ¹H NMR(CDCl₃): δ 3.82 (3H, s, 4-OMe), 5.50 (1H, d, J=2.2 Hz, H-3), 5.95 (1H,d, J=2.2 Hz, H-5), 6.58 (1H, d, J=16.2 Hz, H-7), 7.33 (1H, br. t, J=7.1Hz, 4′-H), 7.38 (2H, br. t, J=7.1 Hz, 3′, 5′-H), 7.49 (2H, br. d, J=7.1Hz, 2′, 6′-H), 7.50 (1H, d, J=16.2 Hz, H-8). 150 MHz ¹³C NMR (CDCl₃): δ55.9 (q, 4-OMe), 88.8 (d, C-3), 101.3 (d, C-5), 118.6 (d, C-7), 127.4(d, C-2′ and C-6′), 128.9 (d, C-3′ and C-5′), 129.4 (d, C-4′), 135.2 (s,C-1′), 135.7 (d, C-8), 158.6 (s, C-6), 164.0 (s, C-2), 171.0 (s, C-4).

The structural formula thereof is shown below.

Compound 2 (KAVA-4) (+)-Dihydrokavain;(4-methoxy-2-(2-phenylvinyl)-2H,3H-oxin-2-one

m.p. 57–60° C., colorless needles [α]_(D) ²⁰+45.7° (c0.50, CHCl₃) EI-MS:m/z 232 (M⁺, 61%), 200 (30%), 173 (15%), 141 (26%), 127 (100%) HR-MS:m/z 232.1094, C₁₄H₁₆O₃ requires 232.1100 FT-IR (KBr) ν_(max) cm⁻¹: 1707(C═O), 1624, 1225, 1090, 1038 UV (EtOH) λ_(max) nm (log ε): 233.0(4.01), 207.0 (4.08)

600 MHz ¹H NMR (CDCl₃): δ 1.93 (1H, m, H-7), 2.13 (1H, m, H-7), 2.30(1H, dd, J=3.8, 17.0 Hz, H-5), 2.50 (1H, ddd, J=1.6, 11.8, 17.0 Hz,H-5), 2.79 (1H, m, H-8), 2.88 (1H, m, H-8), 3.72 (3H, s, 4-OMe), 4.36(1H, m, H-6), 5.14 (1H, d, J=1.6 Hz, H-3), 7.20 (1H, br. t, J=7.1 Hz,4′-H), 7.21 (2H, br. d, J=7.1 Hz, 2′, 6′-H), 7.49 (2H, br. t, J=7.1 Hz,3′, 5′-H). 150 MHz ¹³C NMR (CDCl₃): δ 30.9 (t, C-8), 33.0 (t, C-5), 36.3(t, C-7), 55.9 (q, 4-OMe), 74.7 (d, C-6), 90.3 (d, C-3), 126.1 (d,C-4′), 128.4 (d, C-2′ and C-6′), 128.5 (d, C-3′ and C-5′), 140.8 (s,C-1′), 167.2 (s, C-2), 172.7 (s, C-4).

The structural formula thereof is shown below.

Compound 3 (KAVA-5) (+)-Kavain;(4-methoxy-2-(2-phenylvinyl)-2H,3H-oxin-2-one)

m.p. 106–108° C., colorless needles [α]D_(D) ²⁰+116.3° (c1.01, CHCl₃)EI-MS: m/z 230 (M⁺, 27%), 202 (43%), 186 (13%), 128 (31%), 98 (100%)HR-MS: m/z 230.0951, C₁₄H₁₄O₃ requires 230.0943 FT-IR (KBr) ν_(max)cm⁻¹: 1703 (C═O), 1626, 1248, 1231 UV (EtOH) λ_(max) nm (log ε): 244.5(4.41), 205.0 (4.48) 600 MHz ¹H NMR (CDCl₃): δ 2.54 (1H, dd, J=4.4, 17.0Hz, H-5), 2.66 (1H, ddd, J=1.4, 11.0, 17.0 Hz, H-5), 3.76 (3H, S,4-OMe), 5.05 (1H, ddd, J=4.4, 6.3, 11.0 Hz, H-6), 5.19 (1H, d, J=1.4 Hz,H-3), 6.26 (1H, dd, J=6.3, 15.9 Hz, H-7), 6.73 (1H, br, d, J=15.9 Hz,H-8), 7.27 (1H, br. t, J=7.1, Hz, 4′-H), 7.33 (2H, br. t, J=7.1 Hz, 3′,5′-H), 7.39 (2H, br. d, J=7.1 Hz, 2′, 6′-H). 150 MHz 13C NMR (CDCl₃): δ33.2 (t, C-S), 56.0 (q, 4-OMe), 75.8 (d, C-6), 90.4 (d, C-3), 125.4 (d,C-7), 126.6 (d, C-2′ and C-6′), 128.2 (d, C-4′), 128.6 (d, C-3′ andC-5′), 133.0 (d, C-8), 135.7 (s, C-1′), 166.6 (s, C-2), 172.2 (s, C-4).

The structural formula thereof is shown below.

Compound 4 (KAVA-2) Yangonin;(4-methoxy-6-(2-(4-methoxyphenyl)vinyl)-2H-pyran-2-one)

m.p. 153–155° C., yellow needles EI-MS: m/z 258 (M⁺, 100%), 230 (38%),215 (13%), 187 (33%) HR-MS: m/z 258.0896, C₁₅H₁₄O₄ requires 258.0892FT-IR (KBr) ν_(max) cm⁻¹: 1717 (C═O), 1644 (C═C), 1603, 1555, 1256, 1154UV (EtOH) λ_(max) nm (log ε): 357.5 (4.42), 260.0 (3.89), 218.0 (4.28)600 MHz ¹H NMR (CDCl₃): δ 3.81 (3H, s, 4′-OMe), 3.82 (3H, s, 4-OMe),5.47 (1H, d, J=2.2 Hz, H-3), 5.89 (1H, d, J=2.2 Hz, H-5), 6.44 (1H, d,J=15.7 Hz, H-7), 6.90 (2H, d, J=8.8 Hz, 3′, 5′-H), 7.44 (2H, d, J=8.8Hz, 2′, 6′-H), 7.45 (1H, d, J=15.7 Hz, H-8). 150 MHz ¹³C NMR (CDCl₃): δ55.3 (q, 4′-OMe), 55.8 (q, 4-OMe), 88.3 (d, C-3), 100.4 (d, C-5), 114.3(d, C-3′ and C-5′), 116.3 (d, C-7), 127.9 (s, C-1′), 128.9 (d, C-2′ andC-6′), 135.4 (d, C-8), 159.0 (s, C-6), 160.7 (s, C-4′), 164.1 (s, C-2),171.2 (s, C-4).

The structural formula thereof is shown below.

Compound 5 (KAVA-1) (+)-Methysticin;(2-(2-benzo[3,4-d]1,3-dioxolan-5-ylvinyl-4-methoxy-2H,3H,-oxin-2-one)

m.p. 139–141° C., colorless needles [α]D_(D) ²⁰+115.9° (c0.50, CHCl₃)EI-MS: m/z 274 (M⁺, 100%), 246 (10%), 175 (19%), 148 (81%), 135 (82%)HR-MS: m/z 274.0833, C₁₅H₁₄O₅ requires 274.0841 FT-IR (KBr) ν_(max)cm⁻¹: 1711 (C═O), 1628, 1252, 1217, 1038 UV (EtOH) λ_(max) nm (log ε):305.5 (3.89), 264.5 (4.13), 225.5 (4.38), 207.0 (4.44) 600 MHz ¹H NMR(CDCl₃): δ 2.53 (1H, dd, J=4.4, 17.0 Hz, H-5), 2.65 (1H, ddd, J=1.4,11.0, 17.0 Hz, H-5), 3.77 (3H, s, 4-OMe), 5.02 (1H, ddd, J=4.4, 6.6,11.0 Hz, H.-6), 5.19 (1H, d, J=1.4 Hz, H-3), 6.07 (2H, s, —O—CH₂—O—),6.09 (1H, dd. J=6.6, 15.9 Hz, H-7), 6.64 (1H, br. d, J=15.9 Hz, H-8),6.76 (1H, d, J=8.0 Hz, 5′-H), 6.83 (1H, dd, J=1.9, 8.8 Hz, 6′-H), 6.92(1H, d, J=1.9 Hz, 2′-H). 150 MHz 13C NMR (CDCl₃) δ 33.3 (t, C-5), 56.1(q, 4-OMe), 76.0 (d, C-6), 90.5 (d, C-3), 101.2 (t, —O—CH₂—O—), 105.8(d, C-2′), 108.3 (d, C-5′), 121.7 (d, C-6′), 123.6 (d, C-7), 130.1 (s,C-1′), 132.9 (d, C-8), 147.8 (s, C-4′), 148.1 (s, C-3′), 166.8 (s, C-2),172.3 (s, C-4).

The structural formula thereof is shown below.

Compound 6 (KAVA-6) (+)-Dihydromethysticin (Planta Med., 64, 504–506,1998)

The structural formula thereof is shown below.

Compound 7 7,8-Epoxyyangonin; (4-Methoxy6-[2-(4-methoxyphenyl)oxirane]-2H-pyran-2-one)

[α]D_(D) ¹⁸+13.04° (c1.51,CHCl3) EI-MS: m/z 274 (M⁺, 5%), 258 (100%),230 (64%), 187 (66%) HR-MS: m/z 274.0851, Cl₅H₁₄O₅ requires 274.0841FT-IR (KBr) ν_(max) cm⁻¹: 2940, 1721 (C═O), 1645 (C═C), 1613, 1566,1252, (C—O—C), 1181 600 MHz ¹H NMR (CDCl₃): δ 3.69(3H, s, 4′-OMe),3.77(3H, s, 4-OMe), 5.22(1H, d, J=2.2 Hz, H-3), 5.73(1H, d, J=2.2 Hz,H-5), 6.82(2H, d, J=8.7 Hz, 2′, 6′-H), 7.25(2H, d, J=8.7 Hz, 3′, 5′-H).

150 MHz ¹³C NMR(CDCl₃): δ 42.9(d, C-7), 45.5(d, C-8), 55.1(q, 4′-OMe),55.6(q, 4-OMe), 87.6(d, C-3), 101.2(d, C-5), 113.8(d, C-3′ and C-5′),128.4(d, C-2′ and C-6′), 129.4(s, C-1′), 158.5(s, C-4′), 162.9(s, C-6),164.1(s, C-2), 107.5(s, C-4).

The structural formula thereof is shown below.

Test Examples in relation to the present invention will next bedescribed.

Test Example 1 Inhibition of TNF-α Production (In Vitro)

In response to stimulation by okadaic acid(9,10-deepithio-9,10-didehydroacanthifolicin), BALB/3T3 cells produceTNF-α. The compounds of the present invention were investigated in termsof inhibitory effect on TNF-α production.

An MEM medium (product of Nissui) containing 10% fetal calf serum(product of Biocell Laboratory) was injected into 12-well multiplates(product of Corning), and BALB/3T3 cells were disseminated at 2×10⁵cells/well. The cells were cultured in a carbon dioxide gas incubator(5% Co₂, humidified, 37° C.). Subsequently, KAVA-1, KAVA-2, KAVA-3,KAVA-4, or KAVA-5 was added to the wells at a concentration shown inFIG. 1, and the cells were cultured for one hour. No KAVA compound wasadded to the control wells. After completion of culturing, okadaic acid(carcinogenisis promoter isolated from Halichondria okadai) was added toeach well at a final concentration of 0.2 μM, and culturing wasperformed for 24 hours. After completion of this culturing, the TNF-αconcentration of the supernatant of each well was measured by means ofELISA system (product of Genzyme). The results are shown in FIG. 1. InFIG. 1, the amount of TNF-α release corresponding to each compoundconcentration is represented by a percent concentration based on theamount of TNF-α release measured for the control (100%).

KAVA-3 was found to inhibit TNF-A production to approximately 60% (at 10μM) the TNF-α production of the control; to approximately 22% (at 50 μM)the TNF-α production of the control; and to approximately 0% (completeinhibition) (at 100 μM). KAVA-2 was found to inhibit TNF-α production toapproximately 22% (at 100 μM). KAVA-5 and KAVA-1 exerted similarinhibitory effects; i.e., exerted inhibition to approximately 39% (at100 μM). KAVA-4 exerted no inhibitory effect at concentrations of 50 μMor less, but exerted inhibition to approximately 60% (at 100 μM). Inother words, KAVA-3 and KAVA-2 exerted a strong TNF-A productioninhibitory effect, and KAVA-5 exerted a TNF-α inhibitory effect to anextent similar to that of KAVA-1.

Test Example 2 TNF-α Inhibitory Effect (In Vivo)

Male BALB/cAnNCrj mice of 6 weeks age were purchased from Japan CharlesRiver, and those having body weights of 30 g or lower were tested.

Seven mice groups, each group consisting of six mice, were provided;i.e., 1) a group to which distilled water for injection was administered(non-treated group) (N); 2) a group to which a 0.3% carboxymethylcellulose-Na (0.3% CMC-Na) suspension was administered (control group)(C); and 3) five groups to which KAVA-1, KAVA-2, KAVA-3, KAVA-4, andKAVA-5, respectively, were administered (0.3% CMC-Na was used as asolvent).

Each of KAVA-1, KAVA-2, KAVA-3, KAVA-4, and KAVA-5 was prepared to adrug liquid of 40 mg/10 ml. The liquid was intraperitoneallyadministered at 10 ml/kg (dose: 40 mg/kg), and 0.3% CMC-Na wasintraperitoneally administered at 10 ml/kg to each corresponding group.Lipo-polysaccharide (LPS) (product of SIGMA) was dissolved inphysiological saline, and the solution was intraperitoneallyadministered to each mouse in an amount of 0.2 ml (50 μg/mouse)immediately after administration of the drug liquid.

After 90 minutes from administration of LPS, blood was collected fromthe eye socket, and the collected sample was allowed to stand for onehour at room temperature. Subsequently, the sample was centrifuged at11,000 rpm for five minutes, and the serum was collected. The TNF-α inthe serum was assayed by use of an ELISA kit ([(m)TNFα] mouse ELISAsystem, product of Amersham Pharmacia Biotech K.K.). The results areshown in FIG. 2.

The results shown in FIG. 2 indicate that KAVA-4 significantly (p<0.5)inhibited TNF-α production as compared with the control group (C), andthe TNF-α production inhibitory effect exerted by KAVA-1 was almostcomparable to that exerted by KAVA-2.

As is clear from the above test results, all kavalactones of the presentinvention (compounds 1 to 5) can inhibit TNF-α production. The TNF-αproduction inhibitory effect of compounds 6 and 7 can also be confirmedon the basis of the above test results.

Since the TNF-α production inhibitor of the present invention and thepreventive, ameliorating, or therapeutic agent of the present inventionfor a variety of diseases caused by abnormal production of TNF-α arehighly safe and exhibit an excellent effect of inhibiting TNF-αproduction, the inhibitor and agent are useful as a preventive,ameliorating, or therapeutic agent for mammals, including humans, andfor a variety of diseases caused by abnormal production of TNF-α,including cachexia attributed to cancer or infectious diseases, septicshock, chronic rheumatoid arthritis, inflammatory diseases such asulcerative colitis and Crohn disease, osteoarthritis, Kawasaki'sdisease, multiple sclerosis, Behchet's disease, systemic lupuserythematosus (SLE), rejection during bone marrow transplantation,multiple organ failure, malaria, AIDS, meningitis, hepatitis, andtype-II diabetes.

1. A method of inhibiting TNF-α production in a patient in need thereof,comprising administering to the patient an therapeutically effectivedose of dihydrokavain, wherein said patient suffers from one or morediseases selected from the group consisting of septic shock, chronicrheumatoid arthritis, ulcerative colitis, crohn disease, osteoarthritis,Kawasaki's disease, multiple sclerosis, Behcet's disease, systemic lupuserythematosus, rejection during bone marrow transplantation, multipleorgan failure, malaria, AIDS, meningitis, hepatitis, and type-IIdiabetes.
 2. The method of claim 1, wherein said patient suffers fromchronic rheumatoid arthritis.
 3. The method of claim 1, furthercomprising administering at least one additional kavalactone selectedfrom the group consisting of kavain, yangonin, methysticin,dihydromethysticin, and 7,8-epoxyyangonin.